HIV-1 persistence in viral reservoirs has been the major obstacle for the eradication of HIV-1. Latently infected T cells are one of the potential sources of the virus that is found as residual low-level viremia (LLV) and latent infection of resting memory CD4+ T cells is well demonstrated. Assays have been developed to quantify cells that harbor replication-competent HIV-1 among resting CD4+ T cells in patients on HAART. A viral outgrowth assay is the current gold-standard method to measure latently HIV-1 infected resting CD4+ T cells. However, this assay requires large numbers of resting CD4+ T cells purified from blood and is costly and labor- intensive involving a serial dilution to measure infectious units per million (IUPM) resting CD4+ T cells. To overcome the issues with the current assay, in this application we propose an assay based on next- generation sequencing (NGS) technology. In the proposed assay, the number of different sequences of the viruses bulk-cultured from the resting CD4+ T cells will be directly analyzed to score the number of latently HIV-1-infected CD4+ T cells. Since deep sequencing is highly sensitive to detect multiple variants in a viral population, it will be possible to count the number of distinct viruses that hae been induced to replicate in a bulk culture. These studies will provide valuable information for curing HIV infection and lead to a development of an assay to quantify the latent HIV-1 reservoir with improved accuracy, less time and resource consuming, and less labor intensive.